CN1637152A - Detection reagent for thermostable direct hemolysin-related hemolysin gene of vibrio parahaemolyticus - Google Patents

Detection reagent for thermostable direct hemolysin-related hemolysin gene of vibrio parahaemolyticus Download PDF

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CN1637152A
CN1637152A CNA2004100905255A CN200410090525A CN1637152A CN 1637152 A CN1637152 A CN 1637152A CN A2004100905255 A CNA2004100905255 A CN A2004100905255A CN 200410090525 A CN200410090525 A CN 200410090525A CN 1637152 A CN1637152 A CN 1637152A
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益田升佳
堀江隆一
保川清
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Abstract

A detection reagent for detecting thermostable direct hemolysin-related hemolysin (TRH) gene in an amplification process which specifically amplifies TRH1 and TRH2 RNA, which reagent comprises a first primer having a sequence complementary to a specific sequence of an RNA derived from the TRH gene, and a second primer having a sequence homologous to said specific sequence.

Description

Detection reagent for thermostable direct hemolysin-related hemolysin gene of vibrio parahaemolyticus
Technical Field
The present invention relates to a detection reagent for detecting Vibrio parahaemolyticus (Vibrio parahaemolyticus) in clinical examination, public health, food inspection and food poisoning examination.
Background
Vibrio parahaemolyticus is generally considered to be a pathogenic microorganism that causes infectious food poisoning. In contrast to 95% or more of Vibrio parahaemolyticus isolated from gastroenteritis patients being Kanagawa phenomenon-positive strains which exhibit hemolytic activity on Wagatsuma medium, 99% of strains isolated from fish and water are Kanagawa phenomenon-negative. Therefore, it is considered that there is a close relationship between pathogenic Vibrio parahaemolyticus and Kanagawa's phenomenon.
Subsequently, the occurrence of this kanagawa phenomenon was determined to be caused by the release of Thermostable Direct Hemolysin (TDH) from vibrio parahaemolyticus to the outside of bacterial cells, which led to an increase in the interest of vibrio parahaemolyticus as a pathogenic factor. At present, five types of TDH1 to TDH5 are known as TDH genes.
More recently, from a microbial strain showing pathogenicity despite being negative to Kanagawa phenomenon, a hemolysin having a base sequence similar to TDH and having partial common antigenicity (TDH-related hemolysin [ TRH ]) was identified. At present, two types of TRH1 and TRH2 are known for the TRH gene.
Although a method involving evaluating kanagawa phenomenon after amplification culture or isolation culture to detect and identify vibrio parahaemolyticus is known, a method of detecting a specific sequence present in the vibrio parahaemolyticus gene or RNA derived from the gene after amplification of the sequence is preferable in terms of sensitivity, rapidity and simplicity of detection. A method of amplifying a target nucleic acid at a constant temperature is particularly preferable in terms of automation of the detection system.
A method for detecting and identifying Vibrio parahaemolyticus has been reported in which RNA from the TRH1 or TRH2 gene is specifically amplified at a relatively low temperature (41 ℃) (Japanese unexamined patent publication Nos. 2001-258569 and 2001-340087 and 2001-340088). In this method, an RNA amplification method is used in which a specific RNA sequence derived from the TRH1 or TRH2 gene is used as a template, preparing cDNA by RNA-dependent DNA polymerase using a first primer having a sequence complementary to the specific sequence and a second primer having a sequence homologous to the specific sequence to form a double-stranded RNA-DNA, wherein the first primer or the second primer is added with a promoter sequence of RNA polymerase at the 5' end, degrades RNA in double-stranded RNA-DNA by ribonuclease H, thereby obtaining a single-stranded DNA, synthesizing a double-stranded DNA having a promoter sequence by a DNA-dependent DNA polymerase using the single-stranded DNA as a template, which is capable of transcribing RNA consisting of the aforementioned RNA sequence or a sequence complementary to said RNA sequence, wherein said double stranded DNA produces an RNA transcript in the presence of an RNA polymerase, said RNA transcript serving as a template for subsequent cDNA synthesis by an RNA-dependent DNA polymerase.
However, the foregoing method has the following problems. First, the sensitivity of the method is low. According to Japanese unexamined patent publication No. 2001-340087, only the initial RNA amount of at least 10 is indicated3The TRH1RNA data were detected at the time of copying, but whether the initial RNA amount was less than 10 could be detected3Copying (e.g. 10)2Copy) was not clear. According to Japanese unexamined patent publication No. 2001-340088, only an initial RNA amount of at least 10 is indicated3The TRH2RNA data were detected at the time of copying, but whether the initial RNA amount was less than 10 could be detected3Copying (e.g. 10)2Copy) was not clear.
The second problem is that any reagent capable of detecting both TRH1 and TRH2, which is required for practical use, has never been reported in the prior art. For example, according to Japanese unexamined patent publication No. 2001-340087, it is possible to detect an initial RNA amount of at least 103Reagent of copied TRH1RNA detected no TRH 2. Further, according to Japanese unexamined patent publication No. 2001-340088, it is possible to detect an initial RNA amount of at least 103Reagent of copied TRH2RNA detected no TRH 1.
Third, there is no data about the detected speed. Japanese unexamined patent publication Nos. 2001-340087 and 2001-340088 disclose only electropherograms obtained 30 minutes after the start of the reaction, and the reaction rate is not clear.
Therefore, it is an object of the present invention to provide a TRH RNA detection reagent having excellent sensitivity and speed, which is capable of detecting Vibrio parahaemolyticus TRH1 and TRH 2.
Disclosure of Invention
As a result of intensive studies to develop a more sensitive and faster detection reagent for thermostable direct hemolysin-related hemolysin (TRH) RNA of Vibrio parahaemolyticus, the inventors of the present invention developed a reagent which can detect thermostable direct hemolysin-related hemolysin (TRH) RNA of Vibrio parahaemolyticus within 20 minutes and 10 minutes2Reagents for detecting TRH1 or TRH2 at the amount of starting RNA copied.
The present invention relates to a detection reagent for detecting the TRH gene of Vibrio parahaemolyticus present in a sample, which is used in a detection method using an RNA amplification procedure comprising the steps of:
forming a double-stranded RNA-DNA by generating cDNA using a specific sequence of RNA from the TRH gene, i.e., at least a partial sequence of the RNA, as a template by an RNA-dependent DNA polymerase using a first primer having a sequence complementary to the specific sequence and a second primer having a sequence homologous to the specific sequence, wherein the sequence of the first primer or the second primer is added at its 5' end with a promoter sequence of the RNA polymerase;
degrading the RNA portion of the double-stranded RNA-DNA by ribonuclease H, thereby producing single-stranded DNA; and
synthesizing a double-stranded DNA having the promoter sequence, which is transcribed into an RNA consisting of the specific RNA sequence or a sequence complementary to the specific RNA sequence, by a DNA-dependent DNA polymerase using the single-stranded DNA as a template; wherein,
the double-stranded DNA synthesizes an RNA transcription product in the presence of RNA polymerase, and the RNA transcription product serves as a template for subsequent cDNA synthesis by RNA-dependent DNA polymerase;
the reagent comprises the following components in percentage by weight,
a first primer which is an oligonucleotide consisting of at least 10 consecutive bases of the sequence represented by SEQ ID No.2, or an oligonucleotide capable of specifically binding to the specific sequence by deletion, substitution or addition of one or more nucleotides in an oligonucleotide consisting of at least 10 consecutive bases of the sequence represented by SEQ ID No.2, or an oligonucleotide capable of specifically binding to the specific sequence which is hybridizable with an oligonucleotide consisting of at least 10 consecutive bases of the sequence represented by SEQ ID No.2 under high stringency conditions; and
a second primer which is an oligonucleotide consisting of at least 10 consecutive bases of the sequence represented by SEQ ID No.1, or an oligonucleotide capable of specifically binding to a complementary sequence of the specific sequence after deletion, substitution or addition of one or more nucleotides in the oligonucleotide consisting of at least 10 consecutive bases of the sequence represented by SEQ ID No.1, or an oligonucleotide capable of specifically binding to a complementary sequence of the specific sequence, which is hybridizable with the oligonucleotide consisting of at least 10 consecutive bases of the sequence represented by SEQ ID No.1 under high stringency conditions.
High stringency conditions refer to hybridization conditions such as those set forth in the examples below, in the presence of 60mM Tris, 17mM magnesium chloride, 100-.
Preferably, the first primer can be an oligonucleotide consisting of the sequence shown in SEQ ID No.2, and the second primer can be an oligonucleotide consisting of the sequence shown in SEQ ID No. 1.
Further, when it is intended to detect RNA complementary to an RNA derived from a TRH gene, an oligonucleotide having a sequence complementary to the above-mentioned first primer when the sequence from the 5 'end to the 3' end is inverted should be used as the first primer, and an oligonucleotide having a sequence complementary to the above-mentioned second primer when the sequence from the 5 'end to the 3' end is inverted should be used as the second primer.
Preferably, the RNA amplification procedure is performed in the presence of a cleaving oligonucleotide that cleaves the target RNA at the 5 'end of the specific sequence and has a sequence complementary to a region adjacent to and overlapping with the 5' end of the specific sequence. The oligonucleotide is preferably an oligonucleotide consisting of the sequence shown in SEQ ID No.4, 5 or 6.
More preferably, the above-mentioned RNA amplification procedure is carried out in the presence of an oligonucleotide labeled with an intercalating fluorescent dye, and the detection of thermostable direct hemolysin-related hemolysin of Vibrio parahaemolyticus is carried out by measuring the fluorescence intensity of the reaction solution. Here, the sequence of the oligonucleotide is complementary to at least a part of the sequence of the mRNA transcription product, and in the case where the oligonucleotide is complementarily bound to the RNA transcription product, the fluorescent property of the reaction solution is changed as compared with the case where no complex is formed.
Preferably, the above-mentioned oligonucleotide consists of at least 10 consecutive bases of the sequence shown in SEQ. ID No. 3.
The following provides a detailed description of the present invention.
Brief Description of Drawings
FIG. 1 shows the position along the amplification region where each oligonucleotide used in example 1 was located. The numbering of the bases in the figure is given according to the literature (appl. environ. microbiol., 58, 2449-.
FIG. 2(A) shows copying/detecting 10 from 10 in example 27Increase in fluorescence intensity ratio with reaction time at the time of generation of RNA from the initial amount of TRH1RNA copied/detected, (B) display of calibration curve obtained from logarithmic value and rise time of initial amount of RNA. "negative control" refers to a sample in which a diluent is used instead of RNA. An initial RNA quantity of 10 was detectable at a reaction time of about 15 minutes2TRH1RNA at the time of copying/detection, and a correlation between the initial RNA amount and the rise time was also observed.
FIG. 3(A) shows that 10 was copied/detected from 10 in example 27Increase in fluorescence intensity ratio with reaction time at the time of generation of RNA from the initial amount of TRH2RNA copied/detected, (B) display of calibration curve obtained from logarithmic value and rise time of initial amount of RNA. "negative control" refers to a sample in which a diluent is used instead of RNA. An initial RNA quantity of 10 was detectable after a reaction time of about 19 minutes3TRH2RNA at the time of copying/detection, and also observedCorrelation between amount to initial RNA and rise time.
Best mode for carrying out the invention
The following provides a detailed description of the invention.
In the present invention, although the full length base sequences listed in the sequence listing can be used as the first and second primers, since about 10 bases are sufficient for specific binding to a specific nucleic acid sequence or the like, a combination of at least 10 consecutive bases in each sequence can also be used.
The amplification procedure of the present invention includes a NASBA method, a 3SR method or, for example, an RNA detection method (TRC method) described in Japanese unexamined patent publication No. 2000-014400, which amplifies TRH1 and TRH2RNA by the synergistic action of reverse transcriptase and RNA polymerase by reacting them under conditions under which the reverse transcriptase and RNA polymerase can act synergistically. Here, although the temperature is not particularly limited, it is preferably 35 to 50 ℃.
In one aspect of the above-described invention of the present application, the target RNA must be cleaved at the 5' end of the specific sequence. A preferred method of cleaving the target RNA in this manner preferably consists of: the target RNA is cleaved with ribonuclease H or the like by adding an oligonucleotide (cleaving oligonucleotide) having a sequence complementary to a region adjacent to and overlapping with the 5' -end of the specific sequence. The oligonucleotide is preferably an oligonucleotide consisting of the sequence shown in SEQ ID No.4, 5 or 6. In order to inhibit extension reactions at the 3 'end, the 3' terminal hydroxyl group in the cleaving oligonucleotide is preferably chemically modified, e.g., aminated.
Although the amplification product obtained in the above-mentioned nucleic acid amplification method can be detected by a known nucleic acid detection method, in a preferred aspect of this method, the above-mentioned nucleic acid amplification is preferably carried out in the presence of an oligonucleotide labeled with an intercalating fluorescent dye, and then the change in the fluorescent property of the reaction solution is measured. In the oligonucleotide, an intercalating fluorescent dye is bound to the phosphorus atom of the oligonucleotide via a linker, and thus the chimera agent that forms a double strand with the target Nucleic Acid (complementary Nucleic Acid) is partially intercalated into the double-stranded portion, resulting in a change in the fluorescent properties, which leads to the feature that the method does not require isolation for analysis (Ishiguro, T.et al. (1996) Nucleic Acid Res.24(24) 4992-4997).
The sequence bound by the oligonucleotide may be any sequence specific to the TRH RNA, and although not particularly limited thereto, a sequence consisting of at least 10 consecutive bases in the sequence shown in seq.id No.3 or a complementary sequence thereof is preferable. In addition, the hydroxyl group at the 3' end of the oligonucleotide is preferably chemically modified (e.g., by the addition of glycolic acid) to inhibit extension reactions that may occur by using this oligonucleotide as a primer.
Thus, TRH1 and TRH2RNA of Vibrio parahaemolyticus can be amplified and detected rapidly and with high sensitivity in a single tube using a single step at a constant temperature, thereby facilitating automated application.
Although the invention of the present application is explained in more detail below by way of examples, the present invention is not limited to these examples.
Examples
Example 1
The differences in the amplification efficiency of TRH RNA of Vibrio parahaemolyticus of the combinations (a) to (j) shown in Table 1 and FIG. 1 were compared.
(1) A standard RNA sample (616 bases) containing bases 1 to 610 of the RNAs of Vibrio parahaemolyticus TRH1 and TRH2 (the base numbers of the RNAs are referred to Nishibuchi, et al, Appl environ. Microbiol., 58, 2449 and 2457(1992)) was quantified by ultraviolet absorption at 260nm, and then diluted to 10 ℃ with an RNA diluent (10mM Tris-HCl buffer (pH8.0), 1mM EDTA, 5mM DTT, 0.5U/. mu.l RNase inhibitor (Takara Bio)))3Copy/5. mu.l. The control group (negative control) used only diluent.
(2) Mu.l of the reaction solution having the following composition was dispensed into 0.5ml PCR tubes (GeneAmp thin-walled reaction tubes, Applied biosystems), and 5. mu.l of the above-mentioned RNA sample was added to the tubes. In addition, a solution was prepared to combine the first primer, the second primer and the cleaving oligonucleotide in the manner shown in Table 1.
Composition of reaction solution
(the concentrations indicated are in a final reaction solution volume of 30. mu.l)
60mM Tris-HCl buffer (pH 8.6)
17mM magnesium chloride
130mM potassium chloride (except for 100mM in combination (j))
6U RNase inhibitor
1mM DTT
0.25mM each of dATP, dCTP, dGTP and dTTP
3.6mM ITP
ATP, CTP, GTP and UTP each 3.0mM
0.16. mu.M cleaving oligonucleotide
1.0. mu.M second primer
1.0. mu.M first primer
25nM oligonucleotide labeled with intercalating dye (YO-TRH-S-G, SEQ ID No. 3; labeled with intercalating fluorescent dye between "C" at position 5 from 5 'end and "A" at position 6, hydroxyl group at 3' end modified with ethylene glycol group)
13％DMSO
Distilled water for volume adjustment
(3) After the above reaction solution was incubated at 44 ℃ for 5 minutes, 5. mu.l of an enzyme solution having the composition shown below and preheated at 44 ℃ for 2 minutes was added.
Enzyme solution composition (values shown represent values in a final reaction solution volume of 30. mu.l)
2.0% sorbitol
3.6 μ g bovine serum albumin
142U T7 RNA polymerase (Invitrogen)
6.4U AMV reverse transcriptase (Takara Bio)
Distilled water for volume adjustment
(4) Subsequently, the reaction solution in each PCR tube was measured with time at an excitation wavelength of 470nm and a fluorescence wavelength of 520nm using a fluorescence spectrophotometer equipped with a temperature control function and capable of directly measuring the tube while incubating at 44 ℃.
(5) Table 2 shows the "rise time" results obtained for each oligonucleotide combination (the time required for the fluorescence ratio to increase to 1.2 times the sum of the mean of the negative control samples plus 3 standard deviations). Since TRH1 and TRH2RNA were detected within 15 minutes regardless of the use of any of the combinations (a) to (j), it was revealed that the oligonucleotides used in these combinations were effective for detecting TRHRNA of Vibrio parahaemolyticus.
TABLE 1
Combination of Shearing oligonucleotides Second primer First primer Amplification product Length (base)
(a) TR-S22 TR-F23 TR-R21 202
(b) TR-S22 TR-F23 TR-R24 205
(c) TR-S22 TR-F23 TR-R27 208
(d) TR-S26 TR-F26 TR-R21 202
(e) TR-S26 TR-F26 TR-R24 205
(f) TR-S26 TR-F26 TR-R27 208
(g) TR-S30 TR-F29 TR-R21 202
(h) TR-S30 TR-F29 TR-R24 205
(i) TR-S30 TR-F29 TR-R27 208
(j) TR-S26 TR-F26 TR-R20+11 212
Table 1 shows the combinations of first primer, second primer and cleaving oligonucleotide used in the experimental system, and the length of the specific band amplified using these combinations. For each oligonucleotide combination, FIG. 1 shows the localization and amplified regions of the oligonucleotides in the TRH RNA of Vibrio parahaemolyticus. At the 3' end of the cleavage oligonucleotide base sequence, the hydroxyl group is aminated. The region from the "A" at position 1 to the "A" at position 22 from the 5' end in the base sequence of the second primer is the T7 promoter region, and the subsequent region from the "G" at position 23 to the "A" at position 28 is an enhancer sequence.
Cleavage of the oligonucleotide:
TR-S22 (SEQ. ID No.4, base Nos. 33 to 54)
TR-S26 (SEQ. ID No.5, base Nos. 29 to 54)
TR-S30 (SEQ. ID No.6, base Nos. 25 to 54)
Second primer
TR-F23 (SEQ. ID No.7, base Nos. 50 to 72)
TR-F26 (SEQ. ID No.8, base Nos. 50 to 75)
TR-F29 (SEQ. ID No.9, base Nos. 50 to 78)
First primer
TR-R21 (SEQ. ID No.10, base No. 225 to 245)
TR-R24 (SEQ. ID No.11, base Nos. 225 to 248)
TR-R27 (SEQ. ID No.2, base Nos. 225 to 251)
TR-R20+11 (SEQ. ID No.12, base Nos. 236 to 255)
TABLE 2
Combination of Rise time (minutes)
Trh1 103Copy/test Trh2 103Copy/test
(a) 8.9 8.9 8.7 11.3 11.4 11.4
(b) 8.6 8.9 8.8 11.2 11.4 11.5
(c) 9.3 9.0 9.5 12.5 13.3 13.5
(d) 8.8 8.9 8.8 11.0 10.7 11.2
(e) 8.9 8.9 8.3 11.0 10.8 11.4
(f) 9.0 8.9 8.7 11.4 11.4 10.8
(g) 10.0 9.7 9.9 12.4 12.9 12.2
(h) 10.0 9.9 10.1 11.9 12.5 13.7
(i) 10.1 10.0 10.6 13.7 12.6 12.2
(j) 10.8 10.6 11.0 13.2 14.5 13.1
Table 2 shows the oligonucleotide combinations shown in Table 1 at 103Copy/test to determine the results obtained for TRH1 and TRH2 RNA. All of the oligonucleotide combinations shown in table 1 detected TRH1 and TRH2RNA within 15 minutes.
Example 2
Different initial copy numbers of TRH1 and TRH2 RNAs were detected using the combination (j) shown in Table 1.
(1) The Vibrio parahaemolyticus TRH1 and TRH2 RNAs in example 1 were diluted to 10 using an RNA diluent (10mM Tris-HCl (pH8.0), 1mM EDTA, 5mM DTT, 0.5U/. mu.l RNase inhibitor (Takara Bio)) similarly to7Copies/5. mu.l to 10 copies/5. mu.l. The control group (negative control) used only diluent.
(2) Mu.l of the reaction solution having the following composition was dispensed into a PCR tube (volume: 0.5mL, GeneAmp thin-walled reaction tube, Applied Biosystem), and 5. mu.l of the above RNA sample was added.
Composition of reaction solution
(concentrations indicated are in a final reaction solution volume of 30. mu.l)
60mM Tris-HCl buffer (pH 8.6)
17.85mM magnesium chloride
100mM potassium chloride
6U RNase inhibitor
1mM DTT
0.25mM each of dATP, dCTP, dGTP and dTTP
3.6mM ITP
ATP, CTP, GTP and UTP each 3.0mM
0.16. mu.M cleaving oligonucleotide (TR-S26, SEQ. ID No.5, whose hydroxyl group at the 3' terminus was aminated)
mu.M second primer (TR-F26, SEQ. ID No.8)
1.0 μ M first primer (TR-R20+11, SEQ. ID No.12)
25nM oligonucleotide labeled with intercalating dye (YO-TRH-S-G, SEQ ID No. 3; labeled with intercalating fluorescent dye between the "C" at position 5 from the 5 'end and the "A" at position 6, the hydroxyl group at the 3' end being modified with ethylene glycol group)
13％DMSO
Distilled water for volume adjustment
(3) After the above reaction solution was incubated at 44 ℃ for 5 minutes, 5. mu.l of an enzyme solution having the composition shown below and preheated at 44 ℃ for 2 minutes was added.
Enzyme solution composition (values shown represent values for a final reaction solution volume of 30. mu.l)
2.0% sorbitol
3.6 μ g bovine serum albumin
142U T7 RNA polymerase (Invitrogen)
6.4U AMV reverse transcriptase (Takara Bio)
Distilled water for volume adjustment
(4) Subsequently, the reaction solution in each PCR tube was measured with time at an excitation wavelength of 470nm and a fluorescence wavelength of 520nm using a fluorescence spectrophotometer equipped with a temperature control function and capable of directly measuring the tube while incubating at 44 ℃.
FIGS. 2(A) and 3(A) show the change with time of the fluorescence intensity ratio of the sample (fluorescence intensity value at a predetermined time ÷ background fluorescence intensity value) with the time when the time for adding the enzyme is set to 0 minute. In addition, FIGS. 2(B) and 3(B) show the results obtained with respect to the relationship between the logarithmic value of the amount of starting RNA and the "rise time" (the time required for the fluorescence ratio to increase to 1.2 times the sum of the average value of the negative control samples plus 3 standard deviations). Furthermore, the starting RNA amount ranged from 10 copies/detection of 107Copy/detect.
According to FIGS. 2 and 3, 10 was detected in about 15 minutes2Copy TRH1RNA, whereas for TRH2RNA, 10 was detected within about 19 minutes3And (6) copying. Furthermore, since in about 20 minutes, the combination can be detected even in an initial amount of 103The copied/detected TRH1 and TRH2 RNAs allow simultaneous detection of TRH1 and TRH2 RNAs to be performed more quickly and sensitively than in the prior art methods (Japanese unexamined patent publication Nos. 2001 and 340087 and 2001 and 340088).
Industrial applicability
As explained above, the detection method of the present invention facilitates the simultaneous detection of TRH1 and TRH2RNA of Vibrio parahaemolyticus with high sensitivity.
The oligonucleotide of the present invention is not limited to the sequences (having 22 to 30 bases) listed in the sequence listing, but may be a sequence consisting of at least 10 consecutive bases in these sequences. This is because, apparently, a base sequence of about 10 bases is sufficient to ensure the specificity of a primer or probe for a target nucleic acid under relatively low temperature (preferably 44 ℃).
Those skilled in the art will understand that: although the present invention has been described above with reference to specific embodiments and examples, the present invention is not necessarily limited thereto, and many other embodiments, examples and uses, modifications and extensions from these embodiments, examples and uses may be implemented without departing from the scope of the invention of the present application.
Sequence listing
<110> Tosoh Corporation (TOSOH Corporation)
<120> detection reagent for thermostable direct hemolysin-related hemolysin gene of Vibrio parahaemolyticus
<130>1033908
<160>12
<210>1
<211>29
<212>DNA
<213> Artificial sequence
<220>
<223>TR-F29-T7
<400>1
actctacttt gctttcagtt tgctattgg 29
<210>2
<211>27
<212>DNA
<213> Artificial sequence
<220>
<223>TR-R27
<400>2
ttttcttttt atgtttcggt ttgtcca 27
<210>3
<211>27
<212>DNA
<213> Artificial sequence
<220>
<223>YO-TRH-S-G
<400>3
gattcagttt ttattgttgt atttcta 27
<210>4
<211>22
<212>DNA
<213> Artificial sequence
<220>
<223>TR-S22
<400>4
agagttttag tttcataatt aa 22
<210>5
<211>26
<212>DNA
<213> Artificial sequence
<220>
<223>TR-S26
<400>5
agagttttag tttcataatt aatcct 26
<210>6
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<213>TR-S30
<220>
<223>
<400>6
agagttttag tttcataatt aatcctttat 30
<210>7
<211>51
<212>DNA
<213>TR-F23
<220>
<223>
<400>7
aattctaata cgactcacta tagggagaac tctactttgc tttcagtttg c 51
<210>8
<211>54
<212>DNA
<213>TR-F26
<220>
<223>
<400>8
aattctaata cgactcacta tagggagaac tctactttgc tttcagtttg ctat 54
<210>9
<211>57
<212>DNA
<213>TR-F29
<220>
<223>
<400>9
aattctaata cgactcacta tagggagaac tctactttgc tttcagtttg ctattgg 57
<210>10
<211>21
<212>DNA
<213>TR-R21
<220>
<223>
<400>10
ttttatgttt cggtttgtcc a 21
<210>11
<211>24
<212>DNA
<213>TR-R24
<220>
<223>
<400>11
tctttttatg tttcggtttg tcca 24
<210>12
<211>20
<212>DNA
<213>TR-R20+11
<220>
<223>
<400>12
atggttttct ttttatgttt 20

Claims (6)

1. A detection reagent for detecting a thermostable direct hemolysin-related hemolysin (TRH) gene of vibrio parahaemolyticus present in a sample, which is used in a detection method using an RNA amplification procedure comprising the steps of:
forming a double-stranded RNA-DNA by using a specific sequence of an RNA derived from said TRH gene as a template, and producing cDNA by an RNA-dependent DNA polymerase using a first primer having a sequence complementary to said specific sequence and a second primer having a sequence homologous to said specific sequence, wherein the first primer or the second primer has a promoter sequence of the RNA polymerase added to its 5' -end;
degrading the RNA portion of the double-stranded RNA-DNA by ribonuclease H, thereby generating single-stranded DNA; and
synthesizing a double-stranded DNA having the promoter sequence by a DNA-dependent DNA polymerase using the single-stranded DNA as a template, wherein the double-stranded DNA is capable of transcribing an RNA consisting of the specific RNA sequence or a sequence complementary to the specific RNA sequence; wherein,
the double-stranded DNA is subjected to RNA polymerase to produce an RNA transcription product, which serves as a template for the subsequent cDNA synthesis by RNA-dependent DNA polymerase;
the reagent comprises the following components in percentage by weight,
a first primer which is an oligonucleotide consisting of at least 10 consecutive bases of the sequence shown in SEQ ID No.2, or an oligonucleotide capable of specifically binding to the specific sequence by deletion, substitution or addition of one or more nucleotides from the oligonucleotide consisting of at least 10 consecutive bases of the sequence shown in SEQ ID No. 2; and
a second primer which is an oligonucleotide consisting of at least 10 consecutive bases of the sequence shown in SEQ ID No.1, or an oligonucleotide capable of specifically binding to a complementary sequence of said specific sequence by deletion, substitution or addition of one or more nucleotides from the oligonucleotide consisting of at least 10 consecutive bases of the sequence shown in SEQ ID No. 1.
2. The detection reagent according to claim 1, wherein the first primer is an oligonucleotide consisting of a sequence shown in SEQ.ID No. 2.
3. The detection reagent according to claim 1 or 2, wherein the second primer is an oligonucleotide consisting of the sequence shown in SEQ. ID No. 1.
4. The detection reagent according to any one of claims 1 to 3, wherein the RNA amplification process is carried out in the presence of a cleaving oligonucleotide having a sequence complementary to a region adjacent to and overlapping with the 5 'end of the specific sequence, and cleaving the target RNA at the 5' end of the specific sequence; wherein the oligonucleotide is an oligonucleotide consisting of a sequence shown in SEQ ID No.4, 5 or 6.
5. The detection reagent according to any one of claims 1 to 4, wherein the RNA amplification process is carried out in the presence of an oligonucleotide labeled with an intercalating fluorescent dye, and the thermostable direct hemolysin-related hemolysin of Vibrio parahaemolyticus is detected by measuring the fluorescence intensity of the reaction solution; wherein the sequence of said oligonucleotide is complementary to at least a portion of the sequence of an mRNA transcript and wherein the fluorescent properties of the reaction solution are altered in the event of complementary binding of said oligonucleotide to said RNA transcript as compared to the absence of complex formation.
6. The detection reagent according to claim 5, wherein the oligonucleotide labeled with the intercalating fluorescent dye consists of at least 10 consecutive bases of the sequence shown in SEQ. ID No. 3.
CN2004100905255A 2003-09-05 2004-09-03 Detection reagent for thermostable direct hemolysin-related hemolysin gene of vibrio parahaemolyticus Expired - Fee Related CN1637152B (en)

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CN111411161A (en) * 2020-04-02 2020-07-14 深圳市疾病预防控制中心 Primer group and kit for detecting K antigen genotyping of vibrio parahaemolyticus

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WO2014039037A1 (en) * 2012-09-06 2014-03-13 University Of South Carolina Pcr primers for detection of vibrio parahaemolyticus thermostable direct hemolysin (tdh) and tdh-related hemolysin genes
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CN102676459B (en) * 2011-03-09 2014-04-02 上海海洋大学 Monoclonal antibody resistant to thermostable direct hemolysin of vibrio parahaemolyticus and preparation method of monoclonal antibody
CN111411161A (en) * 2020-04-02 2020-07-14 深圳市疾病预防控制中心 Primer group and kit for detecting K antigen genotyping of vibrio parahaemolyticus
CN111411161B (en) * 2020-04-02 2021-02-23 深圳市疾病预防控制中心 Primer group and kit for detecting K antigen genotyping of vibrio parahaemolyticus

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EP1512753B1 (en) 2007-04-18
EP1512753A1 (en) 2005-03-09
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US7495094B2 (en) 2009-02-24

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